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The metabolism of malignant cells differs significantly from that of healthy cells and thus, it is possible to perform metabolic imaging to reveal not only the exact location of a tumor, but also intratumoral areas of high metabolic activity. Herein, we demonstrate the feasibility of metabolic tumor imaging using signal-enhanced 1-13C-pyruvate-d3, which is rapidly enhanced via para-hydrogen, and thus, the signal is amplified by several orders of magnitudes in less than a minute. Using as a model, human melanoma xenografts injected with signal-enhanced 1-13C-pyruvate-d3, we show that the conversion of pyruvate into lactate can be monitored along with its kinetics, which could pave the way for rapidly detecting and monitoring changes in tumor metabolism.

The metabolism of malignant cells differs significantly from that of healthy cells and thus, it is possible to perform metabolic imaging to reveal not only the exact location of a tumor, but also intratumoral areas of high metabolic activity. Herein, we demonstrate the feasibility of metabolic tumor imaging using signal-enhanced 1

COVID-19 is a viral prothrombotic respiratory infection. Heparins exert antithrombotic and anti-inflammatory effects, and might have antiviral properties. We aimed to investigate whether thromboprophylaxis with enoxaparin would prevent untoward hospitalisation and death in symptomatic, but clinically stable outpatients with COVID-19.OVID was a randomised, open-label, parallel-group, investigator-initiated, phase 3 trial and was done at eight centres in Switzerland and Germany. Outpatients aged 50 years or older with acute COVID-19 were eligible if they presented with respiratory symptoms or body temperature higher than 37·5°C. Eligible participants underwent block-stratified randomisation (by age group 50-70 vs70 years and by study centre) in a 1:1 ratio to receive either subcutaneous enoxaparin 40 mg once daily for 14 days versus standard of care (no thromboprophylaxis). The primary outcome was a composite of any untoward hospitalisation and all-cause death within 30 days of randomisation. Analysis of the efficacy outcomes was done in the intention-to-treat population. The primary safety outcome was major bleeding. The study was registered in ClinicalTrials.gov (NCT04400799) and has been completed.At the predefined formal interim analysis for efficacy (50% of total study population), the independent Data Safety Monitoring Board recommended early termination of the trial on the basis of predefined statistical criteria having considered the very low probability of showing superiority of thromboprophylaxis with enoxaparin for the primary outcome under the initial study design assumptions. Between Aug 15, 2020, and Jan 14, 2022, from 3319 participants prescreened, 472 were included in the intention-to-treat population and randomly assigned to receive enoxaparin (n=234) or standard of care (n=238). The median age was 57 years (IQR 53-62) and 217 (46%) were women. The 30-day risk of the primary outcome was similar in participants allocated to receive enoxaparin and in controls (8 [3%] of 234 vs 8 [3%] of 238; adjusted relative risk 0·98; 95% CI 0·37-2·56; p=0·96). All hospitalisations were related to COVID-19. No deaths were reported during the study. No major bleeding events were recorded. Eight serious adverse events were recorded in the enoxaparin group versus nine in the control group.These findings suggest thromboprophylaxis with enoxaparin does not reduce early hospitalisations and deaths among outpatients with symptomatic COVID-19. Futility of the treatment under the initial study design assumptions could not be conclusively assessed owing to under-representation of older patients and consequent low event rates.SNSF (National Research Programme COVID-19 NRP78: 198352), University Hospital Zurich, University of Zurich, Dr-Ing Georg Pollert (Berlin), Johanna Dürmüller-Bol Foundation.

BACKGROUND: Current guidelines recommend a risk-adjusted treatment strategy for the management of acute pulmonary embolism. This is a particular patient category for whom optimal treatment (anticoagulant treatment, reperfusion strategies, and duration of hospitalisation) is currently unknown. We investigated whether treatment of acute intermediate-risk pulmonary embolism with parenteral anticoagulation for a short period of 72 h, followed by a switch to a direct oral anticoagulant (dabigatran), is effective and safe. METHODS: We did a multinational, multicentre, single-arm, phase 4 trial at 42 hospitals in Austria, Belgium, France, Germany, Italy, Netherlands, Romania, Slovenia, and Spain. Adult patients (aged ≥18 years) with symptomatic intermediate-risk pulmonary embolism, with or without deep-vein thrombosis, were enrolled. Patients received parenteral low-molecular-weight or unfractionated heparin for 72 h after diagnosis of pulmonary embolism before switching to oral dabigatran 150 mg twice per day following a standard clinical assessment. The primary outcome was recurrent symptomatic venous thromboembolism or pulmonary embolism-related death within 6 months. The primary and safety outcomes were assessed in the intention-to-treat population. The study was terminated early, as advised by the data safety and monitoring board, following sample size adaptation after the predefined interim analysis on Dec 18, 2018. This trial is registered with the EU Clinical Trials Register (EudraCT 2015-001830-12) and ClinicalTrials.gov (NCT02596555). FINDINGS: Between Jan 1, 2016, and July 31, 2019, 1418 patients with pulmonary embolism were screened, of whom 402 were enrolled and were included in the intention-to-treat analysis (median age was 69·5 years [IQR 60·0-78·0); 192 [48%] were women and 210 [52%] were men). Median follow-up was 217 days (IQR 210-224) and 370 (92%) patients adhered to the protocol. The primary outcome occurred in seven (2% [upper bound of right-sided 95% CI 3]; p

Recent epidemiological and clinical studies have reported a significantly increased risk for melanoma in people with Parkinson's disease. Because no evidence could be obtained that genetic factors are the reason for the association between these two diseases, we hypothesized that of the three major Parkinson's disease-related proteins-α-synuclein, LRRK2, and Parkin-α-synuclein might be a major link. Our data, presented here, demonstrate that α-synuclein promotes the survival of primary and metastatic melanoma cells, which is the exact opposite of the effect that α-synuclein has on dopaminergic neurons, where its accumulation causes neuronal dysfunction and death. Because this detrimental effect of α-synuclein on neurons can be rescued by the small molecule anle138b, we explored its effect on melanoma cells. We found that treatment with anle138b leads to massive melanoma cell death due to a major dysregulation of autophagy, suggesting that α-synuclein is highly beneficial to advanced melanoma because it ensures that autophagy is maintained at a homeostatic level that promotes and ensures the cell's survival.

Tenascin-C (TNC), overexpressed in invasive growths, has been implicated in progression of melanoma, but the source and function of this molecule are not well defined. We found TNC expression at the front of invading melanoma cells, and that adding TNC to matrices enhances individual melanoma cell migration. As TNC is a multidomain protein, we examined the role of the TNC EGF-like (EGFL) repeats as these activate motogenic signaling cascades. We overexpressed a TNC fragment containing the assembly and EGFL domains of TNC (TNCEGFL). TNCEGFL-expressing melanoma cells had lower speed and persistence in 2D migration assays due to a shift in the adhesion-contractility balance, as expression of TNCEGFL delayed melanoma cell attachment and spreading. The less adhesive phenotype was due, in part, to increased Rho-associated kinase (ROCK) signaling concomitant with myosin light chain 2 and MYPT phosphorylation. Inhibition of ROCK activity, which drives transcellular contractility, restored adhesion of TNCEGFL-expressing melanoma cells and increased their migration in 2D. In contrast to the diminished migration in 2D, TNCEGFL-expressing melanoma cells had higher invasive potential in Matrigel invasion assays, with cells expressing TNCEGFL having amoeboid morphology. Our findings suggest that melanoma-derived TNCEGFL exert a role in melanoma invasion by modulating ROCK signaling and cell migration.

The Parkinson disease-associated kinase Pink1 is targeted to mitochondria where it is thought to regulate mitochondrial quality control by promoting the selective autophagic removal of dysfunctional mitochondria. Nevertheless, the targeting mode of Pink1 and its submitochondrial localization are still not conclusively resolved. The aim of this study was to dissect the mitochondrial import pathway of Pink1 by use of a highly sensitive in vitro assay. Mutational analysis of the Pink1 sequence revealed that its N terminus acts as a genuine matrix localization sequence that mediates the initial membrane potential (Δψ)-dependent targeting of the Pink1 precursor to the inner mitochondrial membrane, but it is dispensable for Pink1 import or processing. A hydrophobic segment downstream of the signal sequence impeded complete translocation of Pink1 across the mitochondrial inner membrane. Additionally, the C-terminal end of the protein promoted the retention of Pink1 at the outer membrane. Thus, multiple targeting signals featured by the Pink1 sequence result in the final localization of both the full-length protein and its major Δψ-dependent cleavage product to the cytosolic face of the outer mitochondrial membrane. Full-length Pink1 and deletion constructs resembling the natural Pink1 processing product were found to assemble into membrane potential-sensitive high molecular weight protein complexes at the mitochondrial surface and displayed similar cytoprotective effects when expressed in vivo, indicating that both species are functionally relevant.

A major focus of melanoma research continues to be the search for genes/proteins that may be suitable targets for molecular therapy of primary and metastatic melanoma. In line with this effort, the objective of the study presented herein was to determine whether interfering with cell cycle progression and in particular, the expression and function of select cyclin-dependent kinases, would impair the biological features of advanced melanoma. We provide data, which document that unlike nevi and melanoma in situ, primary and metastatic melanomas express high levels of CDK2, CDK1, and CDK5. Furthermore, we present the results of in vitro and preclinical in vivo studies, which demonstrate that treatment with a small-molecule cyclin-dependent kinase inhibitor that selectively blocks the function of CDK2, CDK5, CDK1, and CDK9, leads not only to inhibition of melanoma cell proliferation and apoptosis of melanoma cells, but also impairs the growth of human melanoma xenografts.

Unlike ubiquitination, which targets proteins for degradation, sumoylation modulates protein-protein interactions of target proteins. Although there are multiple E2 enzymes required for ubiquitination, there is only one E2-conjugating enzyme for sumoylation, which is Ubc9. In line with increasing evidence that sumoylation plays an important role in tumorigenesis, we recently demonstrated that Ubc9 is expressed at high levels in advanced melanomas and that blocking expression of Ubc9 sensitizes melanomas to the cytotoxic effects of chemotherapeutic drugs. To determine whether and to what extent Ubc9 is expressed in other malignancies and their normal tissue counterparts, we undertook a detailed analysis of colon, lung, prostate, and breast cancer tissue microarrays. The findings, presented here, document that in primary colon and prostate cancer, Ubc9 expression is increased compared with their normal tissue counterparts, whereas in metastatic breast, prostate, and lung cancer, it is decreased in comparison with their corresponding normal and primary adenocarcinoma tissues. We also provide evidence that Ubc9 expression correlates positively with Dukes' stage and negatively with the Gleason score as well as breast cancer grade and that Ubc9 expression is substantially higher in the luminal than in the nonluminal type of breast cancer.

Gaining insights into the molecular events that govern the progression from melanoma in situ to advanced melanoma, and understanding how the local microenvironment at the melanoma site influences this progression, are two clinically pivotal aspects that to date are largely unexplored. In an effort to identify key regulators of the crosstalk between melanoma cells and the melanoma-skin microenvironment, primary and metastatic human melanoma cells were seeded into skin organ cultures (SOCs), and grown for two weeks. Melanoma cells were recovered from SOCs by laser microdissection and whole-cell tryptic digests analyzed by nanoflow liquid chromatography-tandem mass spectrometry with an LTQ-Orbitrap. The differential protein abundances were calculated by spectral counting, the results of which provides evidence that cell-matrix and cell-adhesion molecules that are upregulated in the presence of these melanoma cells recapitulate proteomic data obtained from comparative analysis of human biopsies of invasive melanoma and a tissue sample of adjacent, non-involved skin. This concordance demonstrates the value of SOCs for conducting proteomic investigations of the melanoma microenvironment.

SummaryMany mitochondrial proteins are synthesized with N-terminal presequences that are removed by specific peptidases. The N-termini of the mature proteins and thus peptidase cleavage sites have only been determined for a small fraction of mitochondrial proteins and yielded a controversial situation for the cleavage site specificity of the major mitochondrial processing peptidase (MPP). We report a global analysis of the N-proteome of yeast mitochondria, revealing the N-termini of 615 different proteins. Significantly more proteins than predicted contained cleavable presequences. We identified the intermediate cleaving peptidase Icp55, which removes an amino acid from a characteristic set of MPP-generated N-termini, solving the controversial situation of MPP specificity and suggesting that Icp55 converts instable intermediates into stable proteins. Our results suggest that Icp55 is critical for stabilization of the mitochondrial proteome and illustrate how the N-proteome can serve as rich source for a systematic analysis of mitochondrial protein targeting, cleavage and turnover.

Melanoma in its advanced stages is resistant not only to chemotherapy but also to radiation treatment. In line with efforts to identify genes that are key regulators of the disease and as such, may prove valuable targets for adjuvant and neo-adjuvant therapy of melanomas, we previously reported the presence of Serial Analysis of Gene Expression (SAGE) tags, corresponding to the Ataxia Telangiectasia Mutated (ATM) gene, in SAGE libraries generated from tissues representing primary and metastatic melanomas. In the present study, we document that ATM is expressed at high levels in advanced-stage melanomas. Given its crucial role in the cellular response to DNA double-strand breaks (DSB), ionizing radiation, and UV damage, we pursued a series of functional studies involving the targeting of ATM by way of RNA interference or an ATM-specific small-molecule inhibitor, followed by exposure of the cells to ionizing radiation or radiation combined with a DNA-intercalating drug, to test the hypothesis that the high-level expression of ATM prevents melanoma cells from undergoing apoptosis in response to DNA DSB-inducing treatments. However, unlike as demonstrated in the case of other malignancies, our findings summarized herein do not point to ATM as a pivotal DNA damage sensor for advanced-stage melanomas, raising the possibility that in these cells, genes other than ATM regulate and control the repair of DNA DSB.

The presequence translocase of the mitochondrial inner membrane (TIM23 complex) mediates the import of preproteins with amino-terminal presequences. To drive matrix translocation the TIM23 complex recruits the presequence translocase-associated motor (PAM) with the matrix heat shock protein 70 (mtHsp70) as central subunit. Activity and localization of mtHsp70 are regulated by four membrane-associated cochaperones: the adaptor protein Tim44, the stimulatory J-complex Pam18/Pam16, and Pam17. It has been proposed that Tim44 serves as molecular platform to localize mtHsp70 and the J-complex at the TIM23 complex, but it is unknown how Pam17 interacts with the translocase. We generated conditional tim44 yeast mutants and selected a mutant allele, which differentially affects the association of PAM modules with TIM23. In tim44-804 mitochondria, the interaction of the J-complex with the TIM23 complex is impaired, whereas unexpectedly the binding of Pam17 is increased. Pam17 interacts with the channel protein Tim23, revealing a new interaction site between TIM23 and PAM. Thus, the motor PAM is composed of functional modules that bind to different sites of the translocase. We suggest that Tim44 is not simply a scaffold for binding of motor subunits but plays a differential role in the recruitment of PAM modules to the inner membrane translocase.

The import of proteins into mitochondria represents an essential process for the survival of eukaryotic cells. Most mitochondrial proteins are synthesized as cytosolic precursor proteins. A complex chain of reactions needs to be followed to achieve a successful transport of these precursors from the cytosol through the double membrane system to their final destination inside the mitochondria. In order to elucidate the details of the translocation process, in vitro import assays have been developed that are based on the incubation of isolated active mitochondria with natural or artificial precursor proteins containing the appropriate targeting information. Using this basic system, most of the protein components of the import machinery have been identified and functionally characterized. However, a detailed definition of the molecular mechanisms requires more specialized assay techniques. Here we describe modifications of the standard in vitro import assay technique that are based on the utilization of large amounts of recombinant preprotein constructs. The application of saturating amounts of substrate preproteins is a prerequisite for the determination of translocation kinetics and energy requirements of the import process. Accumulation of preproteins as membrane-spanning translocation intermediates further provides a basis for the functional and structural characterization of the active translocation machinery.

The Markers and Tissue Resources for Melanoma meeting convened by the Cancer Diagnosis Program, Division of Cancer Treatment and Diagnosis, Specialized Programs of Research Excellence at the Organ Systems Branch of the National Cancer Institute (NCI), and the Melanoma Research Foundation was held in Gaithersburg, MD on October 2005. The meeting reviewed the current status of biomarkers for early- and advanced-stage melanoma and addressed some of the challenges scientists and clinicians face as they unravel the biology of melanoma and try to apply these findings to patient care. Specifically, the participants focused on molecular changes associated with melanoma progression, potential diagnostic and prognostic markers emerging from molecular profiling studies, and new treatment targets for current and future clinical trials. They also highlighted the ongoing challenges about translational research in melanoma, including availability of tissue resources, and summarized the status of nevus and melanoma tissue microarrays, recently developed as a collaborative project between the melanoma research community and the NCI. The meeting report is intended to provide a perspective on emerging scientific approaches in translational research that can enhance the progress in discovery and validation of markers for melanoma. (Cancer Res 2006; 66(22): 10652-7)

The cellular role of Hsp100/Clp chaperones in maintaining protein stability is based on two functional aspects. Under normal growth conditions they represent components of cellular protein quality control machineries that selectively remove damaged or misfolded polypeptides in cooperation with specific proteases. After thermal stress, proteins of the ClpB subfamily have the unique ability to directly resolubilize aggregated polypeptides in concert with Hsp70-type chaperones, leading to the recovery of enzymatic activity. Hsp78, the homolog of the bacterial chaperone ClpB in mitochondria of eukaryotic organisms, participates in both protective activities. Hsp78 is involved in conferring thermotolerance to the mitochondrial compartment but also participates in protein degradation by the matrix protease Pim1. Despite the high sequence conservation between Hsp78 and ClpB, an analysis of the structural properties revealed significant differences. The identified mitochondrial Hsp78s do not contain N-terminal substrate-binding domains. In addition, formation of the oligomeric chaperone complex was more variable as anticipated from the studies with bacterial ClpB. Hsp78 predominantly formed a trimeric complex under in vivo conditions. Hence, mitochondrial Hsp78s form a distinct subgroup of the ClpB chaperone family, exhibiting specific structural and functional properties.

In this study, we generated three SAGE libraries from melanoma tissues. Using bioinformatics tools usually applied to microarray data, we identified several genes, including novel transcripts, which are preferentially expressed in melanoma. SAGE results converged with previous microarray analysis on the importance of intracellular calcium and G-protein signaling, and the Wnt/Frizzled family. We also examined the expression of CD74, which was specifically, albeit not abundantly, expressed in the melanoma libraries using a melanoma progression tissue microarray, and demonstrate that this protein is expressed by melanoma cells but not by benign melanocytes. Many genes involved in intracellular calcium and G-protein signaling were highly expressed in melanoma, results we had observed earlier from microarray studies (Bittner et al., 2000). One of the genes most highly expressed in our melanoma SAGE libraries was a calcium-regulated gene, calpain 3 (p94). Immunohistochemical analysis demonstrated that calpain 3 moves from the nuclei of non-neoplastic cells to the cytoplasm of malignant cells, suggesting activation of this intracellular proteinase. Our SAGE results and the clinical validation data demonstrate how SAGE profiles can highlight specific links between signaling pathways as well as associations with tumor progression. This may provide insights into new genes that may be useful for the diagnosis and therapy of melanoma.

Melanomas of sporadic and familial origin develop in a stepwise fashion in approximately 40-80% of all cases; yet, the genetic events governing the progression from nevocytic precursor lesions to early and advanced-stage melanomas remain largely unknown. In the present study, we provide an analysis of genes that were identified in four recently generated primary and metastatic melanoma Serial Analysis of Gene Expression (SAGE) libraries. In addition to SAGE tags corresponding to transcripts with unknown function, or to unidentified transcripts, known genes were identified that hitherto have not been shown to be expressed or have a function in early and advanced-stage melanomas and/or melanoma precursor lesions. Conducting fluorescence imaging analysis with cyanine dye-conjugated antibodies and oligonucleotides, we established the expression pattern of ATM, HEI10, PKD1, KAI11, IL-10R, and hypothetical protein FLJ11151 in nevus and melanoma specimens.

Microarray profiling is a powerful approach to establish gene expression patterns for different histopathological stages of a malignancy, and at the same time, to identify individual genes that may have important functions in the early and/or advanced stages of a neoplasm. To identify genes that hitherto have not been shown to be expressed or play a role in advanced-stage melanomas, we conducted microarray analyses with RNAs from primary melanoma and melanoma-positive lymph node specimens. Using RT-PCR, quantitative, real-time RT-PCR, and fluorochrome oligonucleotide-based optical imaging, we established the level and pattern of expression of five of the identified known genes [Suppression of Tumorigenicity 13 (ST13), Cystatin 8 (CST-8), Dyskeratosis Congentia 1 (DKC1), Neuroendocrine Secretory Protein 55 (NESP55), Niemann-Pick Disease, type C2 (NP-C2)], and a gene with unknown function (16.7 kD Hypothetical Protein) in benign and atypical nevocytic lesions, advanced-stage melanomas, and melanoma-positive lymph nodes.

Atypical nevi are the precursors and risk markers of melanoma. Apart from persistently monitoring these nevocytic lesions and resecting them at the earliest signs of clinical changes, there is as yet no systemic clinical treatment available to interfere with their progression to melanoma. To explore clinical treatments that might interfere with and possibly prevent atypical nevus progression, a previous study documented that 3 months systemic low-dose interferon-alpha (IFN-alpha) treatment of patients with a clinical history of melanoma and numerous atypical nevi, led to inactivation of the STAT1 and STAT3 transcription factors in atypical nevi. Based upon this finding, we initiated a second study to determine whether systemic low-dose IFN-alpha treatment also impairs the expression of upstream regulators and downstream targets of STAT1 and STAT3 in atypical nevi. Using cyanine dye-conjugated antibodies, fluorescence imaging analysis revealed expression of JAK2, JNK1, AKT1, NF-kappa B, and IFN-alpha/beta receptor in benign and atypical nevi, and early- and advanced-stage melanomas. To determine possible changes in the level of expression of these molecules in atypical nevi, excised before and after 3 months of systemic low-dose IFN-alpha treatment, newly designed optical imaging software was used to quantitate the captured fluorescent hybridization signals on a cell-by-cell basis and across an entire nevus section. The results of this analysis did not provide evidence that systemic low-dose IFN-alpha treatment alters the level of expression of upstream regulators or downstream targets of STAT1 and STAT3.

The pathogenesis of melanoma is beginning to be better understood due to a broad effort in defining clinically and pathohistologically the different stages of the disease. Cell culture and animal models have helped us to understand the dynamics of tumor progression. In recent years the field has begun systematic approaches to dissect the biological basis of melanoma. The molecular pathways and biological processes that are currently under investigation are expected to yield new markers for diagnosis and prognosis of melanoma, which currently solely rely on clinical and histopathological information. We also predict that the biological investigations will yield new target molecules for rational therapy. This review summarizes highlights of some of the most significant findings in recent years in melanoma biology (for additional reviews, see references 1-7 ).

The import of proteins into mitochondria represents an essential process for the survival of eukaryotic cells. Most mitochondrial proteins are synthesized as cytosolic precursor proteins. A complex chain of reactions needs to be followed to achieve a successful transport of these precursors from the cytosol through the double membrane system to their final destination inside the mitochondria. In order to elucidate the details of the translocation process, in vitro import assays have been developed that are based on the incubation of isolated active mitochondria with natural or artificial precursor proteins containing the appropriate targeting information. Using this basic system, most of the protein components of the import machinery have been identified and functionally characterized. However, a detailed definition of the molecular mechanisms requires more specialized assay techniques. Here we describe modifications of the standard in vitro import assay technique that are based on the utilization of large amounts of recombinant preprotein constructs. The application of saturating amounts of substrate preproteins is a prerequisite for the determination of translocation kinetics and energy requirements of the import process. Accumulation of preproteins as membrane-spanning translocation intermediates further provides a basis for the functional and structural characterization of the active translocation machinery.

Light-based imaging has extensive applications for medicine and biology, and recent advances in optical imaging modalities, such as confocal and multi-photon scanning fluorescence microscopy, bioluminescence, optical coherence tomography, and spectral imaging, have opened new avenues for visualizing and recording over time dynamic changes in genetic, developmental, and disease mechanisms that cannot be captured by conventional light microscopy. In the present article, we focus on spectral imaging, and using human melanoma and its precursor lesions as an example, we describe the ability of spectral imaging to detect early-stage disease, capture gene expression profiles in tissue specimens, and visualize gene functions in tumors growing in living animals.

The stages of melanocytic progression are defined as atypical (dysplastic) nevus, melanoma in situ, melanoma in the radial growth phase (RGP), melanoma in the vertical growth phase (VGP), and melanoma in the metastatic growth phase (MGP). Melanoma in situ and RGP melanoma often develop in contiguous association with atypical nevi. This frequently poses a problem with respect to their early detection. Furthermore, unlike cells obtained from VGP and MGP melanomas, cells derived from melanoma in situ and RGP melanoma do not proliferate in vitro. Thus, compared to the late stages of the disease, less information is available regarding genes expressed in the early stages. To determine whether spectral imaging, a recently developed optical imaging technique, can detect melanoma in situ and RGP melanoma arising in melanoma precursor lesions, atypical nevi in patients with a clinical history of melanoma were subjected to noninvasive macroscopic spectral imaging. To determine at what stage in the progression pathway of melanoma genes having important biological functions in VGP and MGP melanomas are activated and expressed, lesions of melanoma in situ were analyzed by immunohistochemistry and in situ hybridization for expression of some of these known molecular and immunologic markers. The present study demonstrates the capability of noninvasive spectral imaging to detect melanoma in situ and RGP melanoma that arise in contiguous association with atypical nevi. Furthermore, the study provides evidence that genes and antigens expressed in VGP and MGP melanoma are also expressed in melanoma in situ. Because of the dark and variegated pigmentation of atypical nevi, melanoma in situ and RGP melanoma that arise in these melanoma precursor lesions are often difficult to recognize and thus frequently go unnoticed. The application of new optical screening techniques for early detection of melanoma and the identification of genes expressed in the early stages of melanoma development are two important avenues in the pursuit of melanoma prevention. The investigations presented here document that macroscopic spectral imaging has the potential to detect melanoma in its early stage of development and that genes essential for the proliferation and cell adhesion of VGP and MGP melanoma are already expressed in melanoma in situ.

Familial Parkinson disease is associated with mutations in α-synuclein (α-syn), a presynaptic protein that has been localized not only to the cytosol, but also to mitochondria. We report here that wild-type α-syn from cell lines, and brain tissue from humans and mice, is present not in mitochondria but rather in mitochondria-associated endoplasmic reticulum (ER) membranes (MAM), a structurally and functionally distinct subdomain of the ER. Remarkably, we found that pathogenic point mutations in human α-syn result in its reduced association with MAM, coincident with a lower degree of apposition of ER with mitochondria, a decrease in MAM function, and an increase in mitochondrial fragmentation compared with wild-type. Although overexpression of wild-type α-syn in mutant α-syn-expressing cells reverted the fragmentation phenotype, neither overexpression of the mitochondrial fusion/MAM-tethering protein MFN2 nor inhibition/ablation of the mitochondrial fission protein DRP1 was able to do so, implying that α-syn operates downstream of the mitochondrial fusion/fission machinery. These novel results indicate that wild-type α-syn localizes to the MAM and modulates mitochondrial morphology, and that these behaviors are impaired by pathogenic mutations in α-syn. We believe that our results have far-reaching implications for both our understanding of α-syn biology and the treatment of synucleinopathies.

BACKGROUND The prognosis of metastatic melanomas to the brain (MBM) is variable with prolonged survival in a subset. It is unclear whether MBM differ from extracranial metastases (EcM) and primary melanomas (PrM). METHODS To study the biology of MBM, histopathologic analysis of tumor blocks from patients' craniotomy samples and whole-genome expression profiling (WGEP) with confirmatory immunohistochemistry were performed. RESULTS High mononuclear infiltrate and low intratumoral hemorrhage were associated with prolonged overall survival (OS). Pathway analysis of WGEP data from 29 such craniotomy tumor blocks demonstrated that several immune-related BioCarta gene sets were associated with prolonged OS. WGEP analysis of MBM in comparison with same-patient EcM and PrM showed that MBM and EcM were similar, but both differ significantly from PrM. Immunohistochemical analysis revealed that peritumoral CD3+ and CD8+ cells were associated with prolonged OS. CONCLUSIONS MBMs are more similar to EcM compared with PrM. Immune infiltrate is a favorable prognostic factor for MBM. Cancer 2013;119:2737–2746. © 2013 American Cancer Society.

To test the hypothesis that induction of endogenous bFGF can lead to angiogenesis in ischaemic skeletal muscle, we studied the expression of bFGF after transposition of a well-vascularized muscle flap onto an ischaemic hindlimb in the rabbit. The results indicated a marked induction of bFGF mRNA throughout the myoblasts of the well-perfused muscle flap but not the myoblasts of the ischaemic muscle. bFGF protein was detected in the muscle flap, particularly in the myoblasts located closest to a newly formed, adjacent interface, and in the interface itself. In contrast, bFGF expression was not induced after transposition of a well-perfused muscle flap onto healthy muscle tissue. These data provide evidence that the juxtaposition of ischaemic skeletal muscle with healthy mesenchymal tissue triggers an increased expression of bFGF in the myoblasts of the well-perfused muscle. This paracrine induction of bFGF, in turn, leads to increased angiogenesis and regeneration of the ischaemic skeletal muscle.

The prognosis of metastatic melanomas to the brain (MBM) is variable with prolonged survival in a subset. It is unclear whether MBM differ from extracranial metastases (EcM) and primary melanomas (PrM).To study the biology of MBM, histopathologic analysis of tumor blocks from patients' craniotomy samples and whole-genome expression profiling (WGEP) with confirmatory immunohistochemistry were performed.High mononuclear infiltrate and low intratumoral hemorrhage were associated with prolonged overall survival (OS). Pathway analysis of WGEP data from 29 such craniotomy tumor blocks demonstrated that several immune-related BioCarta gene sets were associated with prolonged OS. WGEP analysis of MBM in comparison with same-patient EcM and PrM showed that MBM and EcM were similar, but both differ significantly from PrM. Immunohistochemical analysis revealed that peritumoral CD3⁺ and CD8⁺ cells were associated with prolonged OS.MBMs are more similar to EcM compared with PrM. Immune infiltrate is a favorable prognostic factor for MBM.

The importance of mitochondria as oxygen sensors as well as producers of ATP and reactive oxygen species (ROS) has recently become a focal point of cancer research. However, in the case of melanoma, little information is available to what extent cellular bioenergetics processes contribute to the progression of the disease and related to it, whether oxidative phosphorylation (OXPHOS) has a prominent role in advanced melanoma. In this study we demonstrate that compared to melanocytes, metastatic melanoma cells have elevated levels of OXPHOS. Furthermore, treating metastatic melanoma cells with the drug, Elesclomol, which induces cancer cell apoptosis through oxidative stress, we document by way of stable isotope labeling with amino acids in cell culture (SILAC) that proteins participating in OXPHOS are downregulated. We also provide evidence that melanoma cells with high levels of glycolysis are more resistant to Elesclomol. We further show that Elesclomol upregulates hypoxia inducible factor 1-α (HIF-1α), and that prolonged exposure of melanoma cells to this drug leads to selection of melanoma cells with high levels of glycolysis. Taken together, our findings suggest that molecular targeting of OXPHOS may have efficacy for advanced melanoma.

In an attempt to investigate the expression and possible function(s) of bFGF and the four members of the fibroblast growth factor receptor family, FGFR-1 -4, in a mesenchymal-derived cell system, we analyzed normal human myoblasts and rhabdomyosarcomas. RT-PCR analysis revealed expression of both the ligand bFGF and the FGF receptors, FGFR-1, FGFR-2, FGFR-3 and FGFR-4, in myoblasts and rhabdomyosarcomas; with FGFR-3 at comparably lower levels of detection. Antisense oligodeoxynucleotides targeted against the translation start site and splice-donor acceptor sites of human bFGF and FGFR-1 did not exert proliferation-inhibitory effects in either cell type. Thus, unlike previous studies which demonstrated the importance of bFGF and FGFR-1 as major contributors in sustaining,the proliferation of normal and malignant cells of neuroectodermal origin, the data presented here, indicate that cells of mesenchymal origin may rely on additional, if not altogether different, growth factors and growth factor receptors for their proliferation.

To identify genes expressed in normal human melanocytes but not in malignant melanomas, we previously applied subtractive cDNA hybridization combined with PCR amplification, which led to the isolation of several human melanocyte-enriched partial cDNAs. Three of these cDNA clones were used to isolate their corresponding genomic clones. The chromosomal location of each of the three genes encoded by the individual genomic clones was determined by fluorescent in situ hybridization. The first gene mapped to human chromosome 3p14, the second gene to chromosome 19q13.1, and the third to 2p16-14. In addition, RNase A protection and in situ hybridization analyses documented expression of the gene, located on 3p14, in normal human melanocytes but not in malignant melanomas.

A major focus of melanoma research continues to be the search for genes/proteins that may be suitable targets for molecular therapy of primary and metastatic melanoma. In line with this effort, the objective of the study presented herein was to determine whether interfering with cell cycle progression and in particular, the expression and function of select cyclin-dependent kinases, would impair the biological features of advanced melanoma. We provide data, which document that unlike nevi and melanoma in situ, primary and metastatic melanomas express high levels of CDK2, CDK1, and CDK5. Furthermore, we present the results of in vitro and preclinical in vivo studies, which demonstrate that treatment with a small-molecule cyclin-dependent kinase inhibitor that selectively blocks the function of CDK2, CDK5, CDK1, and CDK9, leads not only to inhibition of melanoma cell proliferation and apoptosis of melanoma cells, but also impairs the growth of human melanoma xenografts.

Over the past few years, high-throughput analyses have provided important novel insights into molecular pathways that play a crucial role in the progression from early to advanced melanoma, and at the same time, they have led to the identification of genes that as part of melanoma progression are upregulated in advanced melanoma. In the present study, we provide evidence that Aurora kinases A and B, 2 key regulators of M phase progression, are upregulated to high levels with progression from melanoma in situ to primary and metastatic melanoma and that inhibiting the expression of these 2 genes by RNA interference or blocking their function with an Aurora kinase–specific small-molecule inhibitor severely impairs melanoma cell proliferation and cell cycle progression and induces melanoma cell apoptosis. In addition, we present the results of systemic treatment of human melanoma xenografts with an Aurora kinase small-molecule inhibitor as well as Aurora kinase targeting vectors.

BackgroundParkinson's disease (PD) is an adult-onset movement disorder of largely unknown etiology. We have previously shown that loss-of-function mutations of the mitochondrial protein kinase PINK1 (PTEN induced putative kinase 1) cause the recessive PARK6 variant of PD.Methodology/principal findingsNow we generated a PINK1 deficient mouse and observed several novel phenotypes: A progressive reduction of weight and of locomotor activity selectively for spontaneous movements occurred at old age. As in PD, abnormal dopamine levels in the aged nigrostriatal projection accompanied the reduced movements. Possibly in line with the PARK6 syndrome but in contrast to sporadic PD, a reduced lifespan, dysfunction of brainstem and sympathetic nerves, visible aggregates of alpha-synuclein within Lewy bodies or nigrostriatal neurodegeneration were not present in aged PINK1-deficient mice. However, we demonstrate PINK1 mutant mice to exhibit a progressive reduction in mitochondrial preprotein import correlating with defects of core mitochondrial functions like ATP-generation and respiration. In contrast to the strong effect of PINK1 on mitochondrial dynamics in Drosophila melanogaster and in spite of reduced expression of fission factor Mtp18, we show reduced fission and increased aggregation of mitochondria only under stress in PINK1-deficient mouse neurons.ConclusionThus, aging Pink1(-/-) mice show increasing mitochondrial dysfunction resulting in impaired neural activity similar to PD, in absence of overt neuronal death.

The import of precursor proteins into mitochondria represents a cell biological process that is absolutely required for the survival of an eukaryotic cell. A complex chain of reactions needs to be followed to achieve a successful transport of mitochondrial proteins from the cytosol through the double membrane system to their final destination. In order to elucidate the details of the translocation process, in vitro import assays have been developed that are based on the incubation of isolated active mitochondria with natural or artificial precursor proteins containing the appropriate targeting information. Although most of the protein components of the import machinery have been identified and functionally characterized using this basic system, the definition of the molecular mechanisms requires more specialized assay techniques. Here we describe modifications of the standard in vitro import assay technique that are based on the utilization of recombinant preprotein constructs. The application of saturating amounts of substrate preproteins is a prerequisite for the determination of translocation kinetics and energy requirements of the import process. Accumulation of preproteins as membrane-spanning translocation intermediates further provides a basis for the functional and structural characterization of the active translocation machinery.

The essential mitochondrial Hsp70 (mtHsp70) is required for the import of mitochondrial preproteins into the matrix compartment. The translocation-specific activity of mtHsp70 is coordinated by its interaction with specific partner proteins, forming the import motor complex that provides the energy for unfolding and complete translocation of precursor polypeptide chains. A major biochemical characteristic of Hsp70-type chaperones is their nucleotide-regulated affinity to polypeptide substrates. To study the role of this allosteric regulation in the course of preprotein translocation, we have generated specific mtHsp70 mutations located within or close to the interface between the nucleotide-binding and the substrate-binding domains. Mitochondria isolated from the mtHsp70 mutants displayed severely reduced import efficiencies in vitro. Two of the mutants exhibited strong growth defects in vivo and were significantly impaired in the generation of an inward-directed, ATP-dependent import force on precursor proteins in transit. The biochemical properties of these two mutant proteins were consistent with defects in the transfer of conformational signals to the substrate-binding domain, resulting in a prolonged and enhanced interaction with imported substrate proteins. Furthermore, interference with the allosteric mechanism resulted in defects of translocation-specific partner protein interaction. We conclude that even a partial disruption of the interdomain communication in the mtHsp70 chaperone results in an almost complete breakdown of its translocation-driving properties.

Optical imaging has become a powerful tool for visualizing and tracking, spatially and in real time, the expression and function of individual genes and proteins, and biological processes at large. In this chapter, we review recent advances in optical imaging techniques, discuss optical imaging-based biological and biomedical studies in live cells and organisms, and present examples for the application of optical imaging in the context of molecular investigations of melanoma and its precursor lesions.

Although patients diagnosed with melanoma of

Melanoma, the most aggressive form of skin cancer, which accounts for 75% of all skin cancer-related deaths, continues to rise at an alarming rate worldwide. Despite a favorable cure rate when surgically removed at an early stage, the response rate of patients with metastatic disease to single agent chemotherapy is less than 15%, and biologic therapies are only marginally effective. Given this bleak picture, there is a great need to identify and characterize genes that play an important role in the advanced stages of melanoma and thus, may represent valuable targets for clinical therapy. The cell adhesion molecules N-cadherin, MCAM and beta3 integrin have been suggested to represent melanoma progression markers; yet, little is known as to whether they may constitute therapeutic targets for the disease. To provide information regarding this aspect, we determined by way of whole-genome and tissue microarray analysis, their level of expression concordant with melanoma progression, and via RNA interference and antisense technology, their role in melanoma cell proliferation, migration, and invasion. The results of these studies demonstrate that N-cadherin and beta3 integrin expression correlates with progression to advanced-stage melanoma, whereas expression of MCAM does not. On the other hand, MCAM and beta3 integrin are the two adhesion molecules that play a pivotal role in melanoma cell migration and invasion, and for this reason, may represent valuable targets for melanoma therapy.

Over the past two decades, several known genes have been shown to govern important functions in the development of primary and metastatic melanomas. However, from this limited number of genes, it is not possible to establish detailed molecular profiles for the early and advanced stages of melanoma development. To gain insights into the genetic profile of every stage of the melanoma progression pathway, and to determine to what extent these profiles are similar or distinct, we performed whole-genome expression profiling of tissue specimens representing normal skin, benign and atypical nevi, and early and advanced-stage melanomas. The results of this study provide first-time evidence that significant molecular changes occur distinctly at the border of/transition from melanoma in situ to primary melanoma, and that genes involved in mitotic cell cycle regulation and cell proliferation constitute the two leading categories of genes associated with these changes.

BACKGROUND: Two prominent biological features of the advanced stages of human melanoma are their high degree of vascularity and high-level expression of basic fibroblast growth factor (bFGF) and fibroblast growth factor receptor-1 (FGFR-1). Given these characteristics, human melanoma serves as an ideal model to address an important question regarding the efficacy of angiogenesis-based cancer therapy. To induce tumor growth arrest and regression, does it suffice to block expression of bFGF and/or FGFR-1 in only the melanoma cells, or is it essential to inhibit expression of bFGF and/or FGFR-1 in both the melanoma cells and the melanoma cell-interspersing vasculature? MATERIALS AND METHODS: Primary and metastatic human melanomas, grown as subcutaneous tumors in nude mice, were injected twice a week with vector constructs containing the human tyrosinase promoter and antisense- oriented human bFGF or FGFR-1 cDNA. On alternating days, the bFGF and FGFR-1 antisense-targeted tumors received injections of cyanine fluorochrome-conjugated antibodies to a human melanoma and mouse blood vessel marker. Noninvasive, dynamic fluorescence imaging was used to document the cellular events that took place inside the tumors as the result of blocking expression of bFGF or FGFR-1 in the melanoma cells. RESULTS: In vivo, ex vivo, and in vitro fluorescence imaging of the bFGF and FGFR-1 antisense-targeted tumors demonstrated that inhibiting bFGF and FGFR-1 signaling in only the melanoma cells suffices to inhibit tumor growth due to massive induction of melanoma cell apoptosis. CONCLUSIONS: The investigations presented in this study document that inhibiting expression of bFGF or FGFR-1 in only the melanoma cells is as effective in blocking tumor growth as simultaneously inhibiting bFGF or FGFR-1 synthesis in the melanoma cells and the melanoma cell-interspersing vasculature. Furthermore, blocking expression of bFGF or FGFR-1 in the melanoma cells did not lead to activation or increased production of another angiogenic molecule, suggesting the absence of a "salvage pathway" that can circumvent or rescue the blockage of bFGF/FGFR-1 in the melanoma cells.

Unlike normal melanocytes, primary and metastatic human melanomas express high levels of basic fibroblast growth factor (bFGF) and fibroblast growth factor receptor-1 (FGFR-1) messenger RNA, and expression of these genes is essential in sustaining the proliferation of malignant melanomas in vitro. To determine whether bFGF and FGFR-1 are also required for tumor formation in these cells, liposome-mediated gene transfer was used to deliver episomal vectors containing antisense-oriented bFGF or FGFR-1 cDNAs into human melanomas, grown as subcutaneous tumors in nude mice. The growth of tumors injected with these constructs was completely arrested or the tumors regressed as a result of blocked intratumoral angiogenesis and subsequent necrosis. Thus, inhibition of bFGF/FGFR-1-mediated signaling may open a new avenue for the treatment of advanced-stage melanomas.